Human renal prokallikrein was purified from 100 litres of urine by DEAE-cellulose batch adsorption, immunoaffinity chromatography, Trasylol-Affi-Gel 10 adsorption to remove kallikrein, and salt gradient HPLC at pH 4.2 with a Pharmacia Mono Q column which partially separated prokallikrein from kallikrein and also removed impurities. Although the freeze-dried Trasylol-Affi-Gel filtrate contained about 90% prokallikrein, the freeze-dried fraction from Mono Q chromatography was a 1:1 mixture of kallikrein and prokallikrein. S-Carboxylation of this fraction, followed by dansylation and 6 N HCl hydrolysis, gave spots for Dns-Ala, Dns-Ile, Dns-Ile-Val, and possibly Alpha-Dns-His on two dimensional polyamide plates with 3 solvent systems. Because Ile-Val has been reported to be at the amino terminus of kallikrein, Ala may be the aminoterminal residue of prokallikrein. Human renal kallikrein showed strong adsorption to Phenyl-Sepharose 4B in 1.0-2.0 M sulfate and phosphate solutions and in 5-10 M chloride and acetate solutions. In one experiment, kallikrein from the immunoaffinity adsorption step was adsorbed batchwise to Phenyl-Sepharose from 2.0 M (NH4)2HPO4, and the adsorbent was packed into a column and eluted stepwise with decreasing concentrations of (NH4)2HPO4. The esterase activity was eluted with a 2-fold purification in 98% yield by 1.0 and 0.5 M (NH4)2HPO4. However, when the same kallikrein fraction was adsorbed to a column of Phenyl-Sepharose and a linear gradient of 2.0 M (NH4)2HPO4 to water was applied, all of the esterase activity apeared near the end of the gradient in an apparent yield of 128%. Rat renal kallikrein and prokallikrein were batch-adsorbed to DEAE-cellulose from diluted rat urine and eluted with 0.5 M NaCl 8n 60-80% yield. The eluate contained 37% prokallikrein, not significantly different from 39% found for the starting urine.